Dengue virus (DENV) is one of the most important human pathogen that affects millions of people in tropical areas of the world, and it is becoming increasingly present in non-tropical areas. DENV is transmitted by mosquitoes and can cause Dengue fever (DF) and Dengue hemorrhagic fever (DHF) and Dengue shock syndrome (DSS). Currently there are no vaccines or antivirals available for DENV and there is no immune competent animal model available to study this important disease. Our group was the first to report that DENV inhibits the production of type I interferon (IFN) in infected cells and this inhibition is mediated by the NS2B3 protease and allows for efficient infection by the virus. We then reported that the NS2B3 cleaves the immune adaptor molecule STING in permissive human primary cells. Interestingly, murine STING is uncleavable by the NS2B3 protease, making this innate immunity adaptor molecule a strong restriction factor for DENV infection in mice. Taking advantage of the data and resources that we generated during our first funding period we will further investigate host factors implicated in DENV sensing in infected cells. We will also identify host and viral factors important for immune evasion by DENV in infected cels. Finally we will develop new mouse models to study Dengue disease based on the restriction factor STING, by generating mice knocked in for human STING, which is susceptible to cleavage by the DENV NS2B3 protease and may prove to be more susceptible to DENV infection. If successful, this will constitute a potential immune competent new mouse model for DENV. We will use relevant DENV primary isolates of all four serotypes with known epidemic histories and sequences as well as recombinant DENV with specific mutations in viral innate immune antagonists and in RNA structures important for immune sensing, such as the current NIAID DENV vaccine prototypes. This application has the potential of developing the first immune competent mouse model to study DENV disease. We will also identify important factors that enable DENV infection in human systems, which could be targeted by antiviral drugs that could interfere with the virus-host interaction, such as the cleavage of STING by the DENV protease NS2B3.